The first of two developmental switches in human hemoglobin expression involves the down-regulation or silencing of the embryonic globin genes. We have previously identified a developmental silencer located around 250 bp 5' of the cap site of the epsilon globin gene which contains binding sites for transcription factors, GATA-1 and YY1. This negative regulatory element silences epsilon promoter activity in primary human adult erythroid cells and in adult transgenic mice, and promoter activity is restored by mutations in the GATA-1 and YY1 binding sites. These studies show that GATA-1 acting with other cell specific proteins can participate in both negative and positive globin regulation as co-transfection with a GATA-1 expression vector increases epsilon-globin transcription in K562 cells which express embryonic globins. Further examination of the endogenous epsilon-globin gene in adult erythroid progenitors indicates that epsilon-globin remains active, but at low levels, and produces aberrant transcripts, possibly by transcriptional pausing or inefficient splicing mechanisms. These studies suggest a role for events after transcription initiation in hemoglobin switching and may be possible targets for therapeutic intervention in hemoglobinopathies. Recent examination of other DNA regulatory regions within the 5' flanking region of the epsilon globin gene suggest negative elements located 3 kb and 1.7 kb 5' of the cap site, the latter appears to be erythroid specific. DNase l footprinting reveals several potential protein binding sites in the 3 kb 5' negative element. These results indicate that the regulation of epsilon globin gene expression may require multiple control elements and that changes in completion or processing of RNA transcripts may contribute to the developmental regulation of the hemoglobin phenotype. Recently we have constructed several transgenic mouse lines expressing fragments of the epsilon globin gene and are testing these elements in this transgenic assay.